AUTORADIOGRAPHY

Radioisotopes Isotopes(which have same chemical but different physical properties) having radioactivity. Natural occurrence is rare because of its instability. They emit different radiations such as α,β and γ. The mass of the atomic nuclei can vary slightly (=isotopes) for a particular element although the number of electrons remains constant and all the isotopes have the same chemical properties. The nuclei of radioactive isotopes are unstable and they disintegrate to produce new atoms and, at the same time, give off radiations such as electrons (β rays) or radiations (γ rays). Naturally occurring radioisotopes are rare because of their instability, but radioactive atom can be produced in nuclear reactors by bombardment of stable atoms with high-energy particles.

Detection of disintegration: Electrical Eg: GM counter, Ionisation counter, Gas flow counter Scintillation Eg: scintillation counter Autoradiography Electrical: This depends on the production of ion pairs by the emitted radiation to give an electrical signal that can be amplified and registered: used in Geiger counter, ionisation counter and gas flow counter (ii) Scintillation: Some materials have the property of absorbing energy from the radiation and re-emitting this in the form of visible light. In a scintillation counter these small flashes of light are converted into electrical impulses. Both of these techniques count the pulses of the disintegrating atoms. They are fast and quantitative. (iii) Autoradiography differs from the pulse-counting techniques in several ways. Each crystal of silver halide in the photographic emulsion is an independent detector, insulated from the rest of the emulsion by a capsule of gelatin. Each crystal responds to the charged particle by the formation of a latent (hidden) image that is made permanent by the process of development. The record provided by the photographic emulsion is cumulative and spatially accurate. It provides information on the localisation and distribution of radioactivity within a sample.Thus there is little point on doing autoradiography on a specimen that is homogeneously labelled. Although it can be quantitative, autoradiography is a much slower and more difficult approach

What is autoradiography…? Radiography is the visualisation of the pattern of distribution of radiation. In general, the radiation consists of X-rays, gamma or beta rays, and the recording medium is a photographic film. For classical X-rays, the specimen to be examined is placed between the source of radiation and the film, and the absorption and scattering of radiation by the specimen produces its image on the film. In contrast, in autoradiography the specimen itself is the source of the radiation, which originates from radioactive material incorporated into it autoradiography is a technique using x ray film to visualise molecules or fragments of molecules that have been radiooactively labelled Producing a radiograph by means of the radiation emitted from the specimen

radiation (usually beta) Principle Radiation will hit silver grains in emulsion and expose them Expose to film or emulsion Isotope will emit radiation (usually beta) Biological tissue or a living organism is injected with a radiolabelled substance with the purpose of investigating its distribution in the organism. At the end of experiment thin tissue sections are placed in contact with a photographic emulsion.the ionic radiation will convert the emulsion as dark spots which makes it possible to localize the radioactive compound (or its metabolites) in the section. The distribution of radioactive material may be investigated as a function of time after the injection of the radiolabelled compound (”pulse labelling”). Incubate tissue with radioactive ligand

HISTORY 1867 Uranium salts responsible for blackening of photographic film(Niepce de st.victor) 1896 K2UO2(SO4)2emits radiation affecting a photographic film(Becquerel) 1924 First autoradiogram –distribution of Po in biological material 1943 Autoradiographic demonstration of 131I in tissue sections of thyroid 1946 Liquid photographic emulsion for autoradiography(Belanger and LeBlond) 1950 ’Stripping film’ technique for microscopic autoradiography 1970 …Numerous biological applications… ~1990 Alternatives to film: Imaging plate technology The first autoradiography was obtained accidently around 1867 when a blackening was produced on emulsions of silver chloride and iodide by uranium salts. Such studies and the work of the Curies in 1898 demonstrated autoradiography before, and contributed directly to, the discovery of radioactivity. The development of autoradiography as a biological technique really started to happen after World war II with the development of photographic emulsions and then stripping film made of silver halide

PROCEDURE Living cells are briefly exposed to a ‘pulse’ of a specific radioactive compound. The tissue is left for a variable time. Samples are taken, fixed, and processed for light or electron microscopy. Sections are cut and overlaid with a thin film of photographic emulsion. Left in the dark for days or weeks (while the radioisotope decays). This exposure time depends on the activity of the isotope, the temperature and the background radiation (this will produce with time a contaminating increase in ‘background’ silver grains in the film). The photographic emulsion is developed (as for conventional photography). Counterstaining e.g. with toluidine blue, shows the histological details of the tissue. The staining must be able to penetrate, but not have an adverse affect on the emulsion. Alternatively, pre-staining of the entire block of tissue can be done (e.g. with Osmium on plastic sections coated with stripping film [or dipping emulsion] as in papers by McGeachie and Grounds) before exposure to the photographic emulsion. This avoids the need for individually (post-) staining each slide. It is not necessary to coverslip these slides The position of the silver grains in the sample is observed by light or electron microscopy Note: the grains are in a different plane of focus in the emulsion overlying the tissue section. Often oil with a x100 objective is used for detailed observation with the light microscope. These autoradiographs provide a permanent record. Full details on the batch of emulsion used, dates, exposure time and conditions should be kept for each experiment

In-vivo autoradiography In-vitro autoradiography Types: In-vivo autoradiography In-vitro autoradiography In-vivo autoradiography - receptors are labelled in intact living tissue by systemic administration of the radioligand (Positron Emission Tomography In-vitro autoradiography - slide-mounted tissue sections are incubated with radioligand so that receptors are labelled under very controlled conditions

MICROSCOPIC AUTORADIOGRAPHY Cont… MICROSCOPIC AUTORADIOGRAPHY Microscopic autoradiography: Resolution down to 0.5 μm Localization of tracer at the tissue-or cellular level Microscopic autoradiography–requirements / sources of error Labelled compound must be insoluble in water –or precautions must be taken to avoid diffusion or leaching out of the material The biological material must contain no compounds reducing the AgBr of the emulsion (”chemography”) Avoid exposure under conditions favoring ”latent image fading” autoradiography–factors of importance for resolution Type and energy of the radiation (range) Distance between the radiolabelled component in the object and the radiation-sensitive emulsion Thickness of the object Thickness of the emulsion The exposure period The size of the AgBr-crystals in the emulsion The size of the developed Ag-grains The sensitivity of the emulsion Possible dislocation of the radioactive material before exposure Maintenance of good tissue quality during the autoradiographic procedure (to facilitate subsequent localization) Hypophysis of rat labelled with 3H

Macroscopic autoradiography Macroscopic autoradiography: Resolution down to ∼50 μm Localization of tracer at the organ level Localization on chromatogram or gel Whale body autoradiography

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